阅读说明：本技术 一种糖化血红蛋白全血质控品的制备方法 (Preparation method of glycosylated hemoglobin whole blood quality control product ) 是由 虞啸炫 欧元祝 于 2021-07-13 设计创作，主要内容包括：本发明涉及一种糖化血红蛋白全血质控品的制备方法,包括将混合人全血按不同的血型进行分类,用滤筛进行过滤,最后加入蛋白还原剂即可。本发明适用于糖化血红蛋白能力验证质控品、正确度控制物和标准物质的制备,从而解决临床糖化血红蛋白检测标准化工作的准确性问题。(The invention relates to a preparation method of a glycosylated hemoglobin whole blood quality control product, which comprises the steps of classifying mixed human whole blood according to different blood types, filtering by using a filter screen, and finally adding a protein reducing agent. The method is suitable for preparing the quality control product, the accuracy control product and the standard substance for verifying the glycosylated hemoglobin capability, thereby solving the accuracy problem of the clinical glycosylated hemoglobin detection standardization work.)

1. A preparation method of a glycosylated hemoglobin whole blood quality control product comprises the following steps:

(1) carrying out blood grouping on the collected single EDTA anticoagulated whole blood with different glycated hemoglobin concentration levels, and classifying according to blood types A, B, O and AB;

(2) mixing single blood of the same blood type, and filtering by using a filter screen;

(3) adding a protein reducing agent into the filtered mixed whole blood, and fully and uniformly mixing;

(4) and (3) subpackaging the mixed whole blood quality control product added with the protein reducing agent in small bottles, and freezing and storing for later use.

2. The method of claim 1, wherein: the protein reducing agent in the step (3) is dithiothreitol DTT, and the adding mass concentration is 0.5-1.5%.

3. The method of claim 2, wherein: the addition mass concentration of the dithiothreitol DTT is 1%.

4. The production method according to any one of claims 1 to 3, characterized in that: the concentration range of the prepared glycosylated hemoglobin whole blood quality control product is 4-12%.

5. The production method according to any one of claims 1 to 3, characterized in that: the application in the preparation of glycosylated hemoglobin capability verification quality control substances, accuracy control substances and standard substances.

Technical Field

The invention belongs to the field of clinical medicine, and particularly relates to a preparation method of a glycosylated hemoglobin whole blood quality control product.

Background

Glycated hemoglobin (GHb) is a product of hemoglobin in red blood cells combined with saccharides in serum, and includes HbA1a, HbA1b, and HbA1c, wherein HbA1c is about 70%. GHb is formed by slow, continuous and irreversible glycation reactions, and its content depends on blood glucose concentration and the contact time of blood glucose and hemoglobin, and is not related to blood drawing time, whether the patient is fasting, whether insulin is used, and other factors. Therefore, HbA1c can effectively reflect the blood sugar condition of a diabetic patient in a period before detection, and is a detection index widely used in clinic for evaluating the long-term blood sugar control level of the diabetic patient. At present, the detection of glycated hemoglobin (HbA1c) is already included in the routine diabetes detection project by a plurality of countries, the association for diabetes of the united states (ADA) in 2010 and the World Health Organization (WHO) in 2011 have more than 6.5% of the blood concentration of HbA1c as a new standard for diagnosing diabetes, the chinese type 2 diabetes prevention and treatment guideline (2020 edition) published by the diabetes society of China (CDS) is in a laboratory with strict quality control, and glycated hemoglobin (HbA1c) determined by a standardized detection method can be used as a supplementary diagnosis standard for diabetes, and the diagnosis standard is more than or equal to 6.5%.

In order to ensure the accuracy of the detection result of HbA1c, the detection results of the glycated hemoglobin are mutually recognized between reagents and laboratories, and along with the establishment of the glycated hemoglobin IFCC reference method, the standardization of the glycated hemoglobin determination has been gradually developed in China. At present, the national Weijian Committee clinical test center and Shanghai market clinical test center develop a capacity verification plan (PTP) and a precision verification plan (TVP) of glycosylated hemoglobin, in order to eliminate matrix effect of each reagent and method for detection, the detection capacity of a clinical laboratory is truly and objectively reflected, clinical mixed whole blood is adopted for a capacity verification quality control product and a precision verification control product, and an IFCC primary reference method or a secondary reference method is adopted for valuing the whole blood quality control product for precision verification and capacity verification. However, in clinical practice, it is found that quality control products directly adopting mixed human whole blood can cause hemolysis and clotting to different degrees, thereby influencing the detection in clinical laboratories and causing inaccuracy of detection results; in addition, the mixed human whole blood quality control product can be subjected to repeated freezing and thawing due to subpackaging and transportation, and the repeated freezing and thawing whole blood quality control product can be subjected to repeated freezing and thawing including HbA_1cThe oxidation and other reactions of the protein molecules inside the system lead to a certain change in the spatial structure of the protein and the amount of charges carried, thereby affecting the detection results of part of the detection system.

Disclosure of Invention

In order to solve the problems of the glycated hemoglobin whole blood quality control product in practice, it is an object of the present invention to provide a method for preparing a glycated hemoglobin whole blood quality control product suitable for a capacity verification alignment and an accuracy verification plan.

The invention provides a preparation method of a glycosylated hemoglobin whole blood quality control product, which comprises the following steps:

(1) carrying out blood grouping on the collected single EDTA anticoagulated whole blood with different glycated hemoglobin concentration levels, and classifying according to blood types A, B, O and AB;

(2) mixing single blood of the same blood type, and filtering by using a filter screen;

(3) adding a protein reducing agent into the filtered mixed whole blood, and fully and uniformly mixing;

(4) and (3) subpackaging the mixed whole blood quality control product added with the protein reducing agent in small bottles, and freezing and storing for later use.

The protein reducing agent in the step (3) is dithiothreitol DTT, and the adding mass concentration is 0.5-1.5%, preferably 1%.

The concentration range of the prepared glycosylated hemoglobin whole blood quality control product is 4-12%.

The method is applied to the preparation of a glycosylated hemoglobin capability verification quality control substance, an accuracy control substance and a standard substance.

Advantageous effects

The mixed whole blood is classified according to different blood types, so that the hemolysis phenomenon of the whole blood quality control product in transportation and long-term repeated freeze-thaw preservation can be reduced, the mixed whole blood is filtered by the filter screen, the clot in the mixed whole blood quality control product can be removed, and the influence of hemolysis and the clot on the detection result is reduced. Dithiothreitol DTT is a small-molecule organic reducing agent, is often used for reducing disulfide bonds in proteins as a strong reducing agent, can be used for preventing intra-molecular or intermolecular disulfide bonds of proteins formed between cysteines in the proteins, and protects sulfhydryl groups in the proteins or enzymes from being oxidized and inactivated. DTT can reduce the oxidation reaction of protein molecules including HbA1c caused by repeated freeze thawing to a certain extent.

The additives in the blood quality control product can generate matrix effects (matrix effects) to different degrees on each detection system, but the matrix effects are absolute and relative, and the matrix effects can be considered to be 'no' matrix effects as long as the matrix effects are accepted to be negligible or errors introduced by the matrix effects on a certain detection system are at an acceptable level through methodology comparison. Experiments are carried out in various detection systems which are commonly used in clinic, and the glycosylated hemoglobin whole blood quality control product prepared by the invention has no obvious matrix effect in various detection systems.

The preparation method provided by the invention is suitable for preparing the glycosylated hemoglobin capability verification quality control product, the accuracy control product and the standard substance, thereby solving the accuracy problem of the clinical glycosylated hemoglobin detection standardization work and providing a basis for the mutual recognition and consistency of the glycosylated hemoglobin results.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

Collecting glycated hemoglobin HbA collected in hospital_1cA single EDTA anticoagulant blood group with concentration ranging from 4% to 12% is used for blood group determination with ABO blood group reagent and classified according to blood type A, B, O, AB. Mixing single EDTA anticoagulation blood of the same blood group according to five concentration gradients, and performing initial measurement on the concentration of the mixed blood by using a glycosylated hemoglobin analyzer Premiers Hb 9120. And (4) coarse-filtering the mixed whole blood with different concentrations by using a filter screen with a 40-mesh screen, and filtering out clots in the blood for later use.

DTT was weighed to a stock concentration of 100mg/mL (10% by mass), added to the filtered mixed whole blood at a ratio of 1:9, and the DTT was added at a concentration of 1% (by mass), and mixed well by gentle inversion. The mixed whole blood quality control products with five concentration gradients are subpackaged by 0.5mL per tube by using an EP tube, and are frozen and stored in a refrigerator at the temperature of 70 ℃ below zero for standby.

The subpackaged whole blood quality control products with five concentration levels are refrigerated and transported at 2-8 ℃ to 20 selected Shanghai hospitals for quality control product applicability investigation, and the selected hospital detection systems comprise main glycosylated hemoglobin brands of berle, Tosoh, Aikolai, Whitman, Primers and the like on the market. The survey results were subjected to data statistics with the median of the survey data as a designated value, and dispersion CV% was calculated for 20 laboratories. The results of the applicability study are analyzed in table 1.

TABLE 1 glycated hemoglobin Whole blood quality control product suitability survey data analysis

Analysis of applicability survey data in table 1 shows that the prepared glycosylated hemoglobin whole blood quality control products with five concentration levels have small dispersion degrees in each detection system, and the dispersion degrees are less than 5%, so that the requirements of a glycosylated hemoglobin capability verification quality control product and an accuracy verification control product can be met.